Trout brain neuropeptidome
EMSL Project ID
38001
Abstract
We have a currently funded National Science Foundation (NSF) grant "Modeling the salmonid hypothalamus-pituitary-gonad axis" that is coming to an end and our intent is to renew this project based on the progress we have made on this project. The current project has used computational modeling to characterize the pattern of endocrine control over the annual reproductive cycle in female rainbow trout (a seasonally breeding fish). An experimental treatment group was included that used a shortened, annual photoperiod to disrupt ovarian development. Considerable information on gene expression (at the mRNA level) in the pituitary, ovary, and liver has been collected for the modeling effort. However, the brain has not been examined. The intent of the renewal would be to extend the present work to the level of protein expression to begin to describe biological function, particularly at the level of the brain. An Environmental Molecular Sciences Laboratory (EMSL) Rapid Access grant would be used to obtain proof-of-concept data using EMSL proteomic capabilities to identify and quantify unknown proteins in the rainbow trout brain, specifically the neuropeptidome. Comparisons of the number of endogenous peptides and variations in their quantity between two samples (early and mid-cycle)will be used to establish efficacy of the proteomic methods available. Samples of various tissues are currently archived at the University of Idaho and would be immediately transferred to EMSL-Pacific Northwest National Laboratory(PNNL) for analysis. We will utilize LTQ-Orbitrap to obtain preliminary LC-MS data using frozen, archived brain samples from female rainbow trout. There are publicly available and PNNL-developed tools (e.g. UStags and ProsightPC) for MS/MS-based endogenous peptide identification for analyzing the resulting MS/MS data against the most recent UniProt/SwissProt database (http://www.uniprot.org/). Other rainbow trout specific protein sequence databases (e.g., GRASP, http://grasp.mbb.sfu.ca; TIGR, www.tigr.org) are available too. Quantitative measurements will be accomplished by direct comparison of MS-peak intensities (label-free approach) using PNNL-developed computational tools (e.g. Dante). Differentially abundant proteins will be incorporated onto a target ion list for subsequent online MS/MS using Orbitrap-Velos and sequential collisionally induced dissociation (CID) and electron transfer dissociation (ETD) in an attempt to obtain interesting peptides for which initial MS/MS data has not been (successfully) acquired. This EMSL Rapid Access proposal would be used to obtain preliminary data to support a proteomic approach for identifying and quantifying novel proteins and justify inclusion of EMSL in the NSF proposal work plan.
Project Details
Project type
Limited Scope
Start Date
2009-10-21
End Date
2009-12-21
Status
Closed
Released Data Link
Team
Principal Investigator